Automating Sample Preparation for LCMS with Shimadzu’s

CLAM-2000SERIES

Conventional LCMS sample pretreatment for forensic samples, such as blood, urine, tissues, and oral fluid, typically involves a manual or semi-automatic process that can take many hours. These methods are time-consuming, expensive and vulnerable to error— but they were the only option available to forensic laboratories.

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The Shimadzu

• Fully automates sample

preparation

• Minimizes human error

• Increases laboratory efficiency by

allowing users to directly place

the sample in the system

CLAM-2000SERIES

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The CLAM-2000 series provides a fully

automated sample preparation

module that can be integrated with a

Shimadzu LCMS analyzer. It was the first

system in the world to fully integrate

sample prep automation for LCMS

workflows, from pretreatment of the

sample to chromatographic analysis.

Once sample preparation is complete,

the CLAM-2000 series delivers the

sample to the LC autosampler for

injection, removing the need for any

human intervention.

The CLAM-2000 series can accept up

to 60 samples at a time, making it the

perfect solution for labs of all sizes.

An intuitive, easy-to-use interface

ensures rapid and reliable operations.

Each sample is processed successively

in parallel, allowing the system to

keep up with current LCMS methods.

A fully automated platform helps you

speed up analysis, minimize labor costs

and greatly reduce human error and

contamination.

Sample Injection

13 min 13 min

Sample Injection Sample Injection

LC-MS/MS Analysis12 min

Traditional Sample Preparation> 60 min

CLAM-2000 Series Sample Preparation6 min

LC-MS/MS Analysis12 min

Sample Preparation

6 min

Sample Preparation

6 min

Sample Preparation

6 min

Sample Dispensing30 µL of serum

Reagent Dispensing60 µL of ACN with IS

Shaking150 sec

Filtration120 sec

Seamless Integration of Sample Preparation and LC-MS/MS Analysis

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Fully Automated Sample Preparation & LCMS Analysis of Drugs in Oral Fluid

Using the CLAM-2000 series, we developed a fast, reliable method for oral fluid analysis. To test the method, we integrated the CLAM-2000 series with a Shimadzu Nexera LC system and a Shimadzu LCMS-8050 triple quadrupole mass spectrometer.

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We implemented a linear gradient from 10% to 60% methanol in only

seven minutes. All samples were collected with a Quantisal® swab and

extracted in diluent. Once the samples were loaded into the carousel, all

steps were automatically performed including LCMS analysis. The initial

sample preparation steps with LCMS analysis took eleven minutes. The

sample-to-sample analysis time took seven minutes, which is equal

to the LCMS method time.

The developed method enabled us to perform fully automated sample

preparation, LC separation and MS analysis of 122 drugs and deuterated

internal standards. For this analysis, we achieved percent relative standard

deviations of ~10% or less for all compounds. The results demonstrated

the effectiveness and high reproducibility of this method.

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Analysis of Illicit Drugs in Multiple Tissue Matrices Using the CLAM-2000 Series

The accurate detection of illicit drugs in tissue samples is crucial in forensic science. In many cases, analysis of drugs from various matrices can be vital in determining the cause of death or events that occurred before death.

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Fully automated sample preparation modules like the CLAM-2000 series help improve

the efficiency and accuracy of this type of analysis.

Using the CLAM-2000 series, we developed a method for analyzing drug

concentrations in tissue samples. We analyzed a total of 15 analytes and 4 internal

standards, which are shown in the table to the right. These were run in duplicate in

whole blood, liver, brain, spleen and muscle. Linear ranges for most compounds were

10 ng/ML – 1000 ng/mL with a five-point calibration curve in each matrix. The results

demonstrated very good linearity for all of the compounds.

Target Compounds and Internal Standards

Morphine-d3

Morphine

Hydromorphone

Codeine

6-AM

Hydrocodone-d3

Hydrocodone

7-Aminoclonazepam

Fentanyl

Buprenorphine

Lorazepam

Clonazepam

Nordiazepam

Alprazolam-d5

Aplrazolam

THC-COOH

THC-d3

Delta-9-THC

THC-OH

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Online Sample Pretreatment and LC-MS/MS Analysis for Screening & Quantitation of Illicit Drugs in Urine

The CLAM-2000 series can be used in a variety of forensic applications. One example is analyzing illicit and prescription drugs in forensic samples such as urine and serum.

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We developed a fully automated method of sample pretreatment and

quantitation for illicit drugs in human urine. This method was performed

using the Shimadzu CLAM-2000 series combined with a Shimadzu LCMS

triple quadrupole system. MRM transitions were monitored for 18 illicit

drugs and 14 isotope-labeled internal standards.

The CLAM-2000 series made it possible to automatically prepare and

analyze the samples. Hands-free automation streamlined the entire

process, from reagent mixing and filtration to dispensing solvents and

derivatization. An automated batch-run program was also used to perform

sample pretreatment and analysis simultaneously. The figure on the left

shows the automated workflow we created to test the method.

Typical automated workflow of urine sample via protein crash and adding internal standard (IS) for LC-MS/MS.

Add 20 µL of sample (urine)

MeOH wetting (conditioning)

Add 20 µL of IS

Add 40 µL of ACN-MeOH

Vortex for 60 seconds

Vacuum filter for 90 seconds

Transferring to LC-MS/MS

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Method performance was evaluated based on linearity, accuracy,

specificity and process efficiency. Reliable quantitation accuracy was

obtained for all drugs, except methadone at 20 ng/mL with an accuracy

of 130%. The results of the linearity test were R2 greater than 0.995

for the 18 illicit drugs, with samples ranging from 20 ng/mL to 200 ng/

mL in urine. Process efficiency was between 62~122% for all analytes

except norbuprenorphine, morphine, MAM and methadone.

We also found that the method offers high specificity, as indicated by

the results in the figure to the right. This figure shows the confirmation

criteria for each target, including quantifier MRM peaks and retention

times. The CLAM-2000 series delivered the high-quality results

needed for precise detection of illicit drugs in urine.

Shimadzu’s CLAM-2000 series automates and integrates sample

preparation, increases laboratory efficiency and prevents sample

contamination — all while providing high accuracy and sensitivity.

When paired with an LCMS system, it offers a complete sample

preparation and analysis solution for forensic laboratories.

Total MRM chromatograms of (A) blank urine and (B) spiked urine

with eighteen illicit drugs (200 ng/mL).

To learn more about how Shimadzu can help you optimize your forensic analysis, visit www.InvestigateYourLab.com.

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To learn more about how Shimadzu can help you optimize your forensic analysis, visit

www.InvestigateYourLab.com

7102 Riverwood Drive, Columbia, MD 21046, USA Phone: 800.477.1227 / 410.381.1227

www.ssi.shimadzu.com

For Research Use Only. Not for use in diagnostic procedures.